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Shikonin, purified from the Chinese medicinal herb, Lithospermum erythrorhizon , induces necroptosis in a variety of cancers although, the mechanisms underlying the anticancer activity of shikonin in lung cancer are not fully understood. This study was designed to clarify whether shikonin can cause necroptosis in non-small cell lung cancer (NSCLC) cells and investigate the mechanism of action. Multiplex and caspase 8 assays were used to analyze effect of shikonin on A549 cells while cytometry with annexin V/PI staining and the MTT assay were used to analyze the mode of cell death. Western blot analysis was used to determine the effect of shikonin-induced necroptosis and autophagy. Xenograft and orthotopic models with A549 cells were used to evaluate the anti-tumor effect of shikonin in vivo. A549 cells were treated with shikonin to evaluate the effect on autophagy and necroptosis. Most of the cell death induced by shikonin could be rescued by the specific necroptosis inhibitor necrostatin-1, but not by the general caspase inhibitor Z-VAD-FMK. Tumor growth was significantly reduced in animals treated with shikonin compared to a control group. Shikonin also increased the protein levels of RIP1 in tumor tissues. Autophagy inhibitors including methyladenine (3-MA), ATG5 siRNA, and bafilomycin A enhanced shikonin-induced necroptosis whereas RIP1 siRNA had no effect on the apoptotic potential of shikonin. In conclusion, our data indicated that shikonin treatment induced necroptosis and autophagy in NSCLC cells. In addition, inhibition of shikonin-induced autophagy enhanced necroptosis, suggesting that shikonin could be a novel therapeutic strategy against NSCLC.

Pemetrexed, a multitarget antifolate used to treat malignant mesothelioma and non-small cell lung cancer (NSCLC), has been shown to stimulate autophagy. In this study, we determined whether autophagy could be induced by pemetrexed and simvastatin cotreatment in malignant mesothelioma and NSCLC cells. Furthermore, we determined whether inhibition of autophagy drives apoptosis in malignant mesothelioma and NSCLC cells. Malignant mesothelioma MSTO-211H and A549 NSCLC cells were treated with pemetrexed and simvastatin alone and in combination to evaluate their effect on autophagy and apoptosis. Cotreatment with pemetrexed and simvastatin induced greater caspase-dependent apoptosis and autophagy than either drug alone in malignant mesothelioma and NSCLC cells. 3-Methyladenine (3-MA), ATG5 siRNA, bafilomycin A, and E64D/pepstatin A enhanced the apoptotic potential of pemetrexed and simvastatin, whereas rapamycin and LY294002 attenuated their induction of caspase-dependent apoptosis. Our data indicate that pemetrexed and simvastatin cotreatment augmented apoptosis and autophagy in malignant mesothelioma and NSCLC cells. Inhibition of pemetrexed and simvastatin-induced autophagy was shown to enhance apoptosis, suggesting that this could be a novel therapeutic strategy against malignant mesothelioma and NSCLC.